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| Cat.No.: | CSC-RG0749 |
| Cell Line Description: | This human PTH2-expressing cell line is made in the Chem-1 host, which supports high levels of recombinant PTH2 expression on the cell surface and contains high levels of the promiscuous G protein Galpha15 to couple the receptor to the calcium signaling pathway. Thus, the cell line is an ideal tool for screening for antagonists of interactions between the PTH2 and its ligands. |
| Background: | Parathyroid hormone (PTH) is recognized by three different class B GPCRs; PTH1, PTH2, and PTH3, which couple to Gs to stimulate cAMP production. The PTH1 receptor is found in high levels in the kidney and bone, where it binds PTH and PTHrP to regulate Ca2+ homeostasis. The PTH2 receptor, which binds PTH and the neuropeptide tuberoinfundibular peptide 39 (TIP-39) but not PTHrP, has about 50% amino acid sequence identity to the PTH1 receptor. PTH2 is found in the greatest volume in the nervous system and at a very low density in the kidney and bone. The PTH3 receptor, which has been recently discovered in the zebrafish, has 61% amino acid identity to the zebrafish PTH1 receptor and also seems to share its ligand affinity. Studies with the selective PTH2 receptor agonist, TIP-39, and the distribution of the receptor in the superficial dorsal horn of the spinal cord suggest the receptor may play a role in pain perception. |
| Growth Properties: | Adherent |
| Host Cell: | Chem-1 |
| Cell Line Validation: | 1. Gene expression: qPCR experiments determined specific silencing of PTH2R.2. Protein expression: PTH2R in this cell line has been validated by immunocytochemical staining.3. EC50 for calcium mobilization by TIP-39: ~ 20.3 nM. |
| Application: | Calcium flux assay, ligand binding assays |
| Sub-type: | PTH receptor |
| Propagation: | Complete growth medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x Nonessential amino acids + 10mM HEPES + 1x Pen-Strep + 250μg/mL Genetecin/G-418 Plating medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 10% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-StrepAtmosphere: air, 95%; carbon dioxide (CO2), 5%Temperature: 37 °C |
| Starting Cells From Frozen Cell Stock: | 1. Immediately upon receipt, thaw cells or place cells in liquid nitrogen. Maintain frozen in liquid nitrogen for up to 5 years. 2. Thaw cells rapidly by removing from liquid nitrogen and immediately immersing in a 37°C water bath. Immediately after ice has thawed, sterilize the exterior of the vial with 70% ethanol. Transfer contents of the vial to a T75 flask containing growth media. Place the flask in a humidified incubator at 37°C with 5% CO2. 3. After 8-24 h, all live cells will be attached. Viability of the cells is expected to be 50-80%. At this time, replace media to remove residual DMSO, and return to incubator. |
| Subculturing: | 1. When cells are approximately 80% confluent, passage the cells as follows: Remove media and wash once with HBSS without Ca2+ and Mg2+ (10 mL/T75). Add 0.05% trypsin/0.2 g/L EDTA (1 mL/T75) and place in humidified incubator at 37°C with 5% CO2 until cells begin to round up and detach (5-10 minutes). Gently rap the side of the flask to dislodge the cells. Neutralize trypsin by addition of 4 mL Growth Media per 1 mL trypsin. 2. Cells are typically passaged 1:10 every 3-4 days. Passaging ratio may be varied according to requirements of the investigator. |
| Mycoplasma: | Mycoplasma Status: Negative (MycoAlert Kit) |
| Freeze Medium: | Freezing medium: DMEM with 4.5 g/L glucose + 4mM glutamine + 20% heat-inactivated FBS + 1x NEAA + 10mM HEPES + 1x Pen-Strep + 10% DMSO |
| Storage: | Liquid nitrogen |
| Preservation: | 1. Frozen stocks of cells should be prepared at the earliest passage possible after thawing, as follows: Count detached cells (prepared as in Subculture-Step 1). Centrifuge cells at 200 x g for 5 min. Resuspend cells at 5 x 10^6 cells/mL in Freezing Media (cell densities of 2-10 x 10^6 are also acceptable if necessary). Dispense 1 mL aliquots into cryopreservation vials. Freeze the cells by a controlled rate process, such as in an isopropanol-jacketed container placed at –70°C overnight. Store the vials in liquid nitrogen. 2. Use of cells immediately after thawing is feasible for some cell lines and is being further validated. Some cell lines may need to be passaged at least once after thawing prior to use in calcium flux assays. Cells should be resuspended in Plating Media for plating for calcium assay. |
| Safety Considerations: | The following safety precautions should be observed.1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum.2. No eating, drinking or smoking while handling the stable line.3. Wash hands after handling the stable line and before leaving the lab.4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells.5. All waste should be considered hazardous.6. Dispose of all liquid waste after each experiment and treat with bleach. |
| Ship: | Dry ice |
| Gene Name: | PTH2R parathyroid hormone 2 receptor [ Homo sapiens ] |
| Official Symbol: | PTH2R |
| Synonyms: | PTH2R; parathyroid hormone 2 receptor; parathyroid hormone receptor 2 , PTHR2; PTH2 receptor; parathyroid hormone receptor 2; PTHR2; |
| Gene ID: | 5746 |
| mRNA Refseq: | NM_005048 |
| Protein Refseq: | NP_005039 |
| MIM: | 601469 |
| UniProt ID: | P49190 |
| Chromosome Location: | 2q33 |
| Pathway: | Class B/2 (Secretin family receptors), organism-specific biosystem; G alpha (s) signalling events, organism-specific biosystem; GPCR downstream signaling, organism-specific biosystem; GPCR ligand binding, organism-specific biosystem; GPCRs, Class B Secretin-like, organism-specific biosystem; Neuroactive ligand-receptor interaction, organism-specific biosystem; Neuroactive ligand-receptor interaction, conserved biosystem; |
| Function: | parathyroid hormone receptor activity; receptor activity; signal transducer activity; |
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